Mass spectrometry (MS) is a technique whereby the mass of individual molecules or compounds can be measured with extremely high accuracy. It is a ubiquitous technique with applications in many areas including sport, medicine, airport security and the food industry. Although tandem mass spectrometry (MS/MS) is one of the most powerful analytical tools available for the elucidation of molecular structure, and can identify the number of unsaturated bonds in a molecule, it often lacks the ability to locate the position of unsaturation within molecules. This can be a significant limitation given the variation in physical and chemical properties of a molecule that can arise as a result of variations in the position of unsaturation.
In recent years mass spectrometry has become arguably the most important tool in the quantification and structural characterisation of lipids within biological extracts. By utilizing electrospray ionization tandem mass spectrometry (ESI-MS/MS), the lipid class, carbon chain length and degree of unsaturation of fatty acid components of lipids can be determined. Unsaturated bond position however, has been largely ignored in this kind of analysis which belies the natural diversity in lipid biochemistry. This is of major importance since lipid isomers differing only by the positions of unsaturation can have distinct biological functions.
One method used to identify the position of unsaturation in intact lipids using mass spectrometry is the collision induced dissociation (CID) of carboxylate anions formed upon fragmentation of the parent phospholipid anion in an MS3 experiment. Comparison of the resultant MS3 spectrum with the MS/MS spectrum of the deprotonated free fatty acid can, in some instances, elucidate the double bond position in the bound fatty acid. In practice however, there are several disadvantages associated with such an experiment; (i) it requires an MS3 capable mass spectrometer, (ii) the low energy CID of deprotonated fatty acids are often not structurally diagnostic, e.g., often only dehydration and/or decarboxylation is observed, and (iii) the alternative high energy CID can produce excessive fragmentation making for very complex interpretation in the absence of comparative standards.
The present inventors have previously demonstrated that in-source ozonolysis is an effective tool in determining the position of double bonds in purified lipids or very simple mixtures of mostly saturated lipids. However, the analysis of complex lipid mixtures, particularly those with a high degree of unsaturation, is insensitive and yields highly complex and structurally ambiguous data. The most significant limitation is that ozone induced dissociation of two ionized lipids of different mass can yield fragments of the same mass. Furthermore, low abundance ozone induced fragment ions can be obscured by unoxidised lipid ions.
There is therefore a need for an improved mass spectrometric method whereby the position(s) of unsaturation in a compound can be determined, that at least partially addresses the deficiencies associated with known methods.